The human sequence-specific transcription factor USF is utilized by adenovirus for expression of the major late transcription unit. We have purified two different forms of this transcription factor from HeLa cell nuclei and isolated cDNA clones corresponding to one of these forms. These studies have revealed that the expression of this transcription factor, as well as its activity, may be subject to multiple levels of control. We now propose to isolate cDNA and antibody probes to the other form of USF and carry out a series of experiments which have two major objectives. The first objective will be to determine the cellular function of the USF proteins by investigating where and when these transcription factors are expressed and how their expression is regulated. Second, we would like to gain a better understanding of the molecular mechanisms by which USF stimulates transcription initiation by RNA polymerase II. For this, we propose to: i) dissect within these proteins the various domains which are involved in specific DNA binding, trans-activating function, or interaction with the TATA box factor TFIID and ii) analyze in detail the involvement of USF in the various steps of the transcription reaction, using a combination of physical (footprinting) as well as functional (template challenge) methods of analysis. In addition, we propose to investigate whether the possible interaction of USF with some components of the transcriptional machinery, or with other regulatory proteins, can be unraveled by using the bacterially-produced transcription factor as an affinity matrix.